鸡骨草叶中N-反式对香豆酰酪氨酸的纯化工艺优化
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摘要
目的:建立鸡骨草叶中N-反式对香豆酰酪氨酸的含量测定方法,并优化其纯化工艺。方法:采用高效液相色谱法测定鸡骨草叶中N-反式对香豆酰酪氨酸的含量,色谱柱为Hypersil BDS C18,流动相为0.1%甲酸水溶液(A)-甲醇(B)(梯度洗脱),流速为1.0 mL/min,柱温为25℃,检测波长为300 nm,进样量为10μL。以聚酰胺树脂为材料,以N-反式对香豆酰酪氨酸得率为指标,采用单因素试验优化N-反式对香豆酰酪氨酸纯化的样品溶液质量浓度、上样量、静置吸附时间等条件。结果:N-反式对香豆酰酪氨酸检测进样量线性范围为2.575～51.50μg(r=0.999 9);定量限为0.000 618μg,检测限为0.000 129μg;精密度、稳定性、重复性试验的RSD均小于3%;加样回收率为97.04%～102.43%(RSD=2.06%,n=6)。最优纯化工艺为样品溶液质量浓度为0.04 g/mL(以鸡骨草叶药材计),上样量为50 m L,上样流速为1.0 m L/min,静置吸附时间为20 min,以水(含0.1%乙酸)、20%乙醇(含0.1%乙酸)、氨水(pH 10)为洗脱溶剂依次洗脱。此纯化工艺条件下,制得的N-反式对香豆酰酪氨酸的平均得率为98.94%,平均干膏含量为61.17 mg/g,平均干膏纯度为19.73%。结论:所建含量测定方法简便、准确、稳定性较好;优化所得工艺稳定、可行。
OBJECTIVE:To establish a method for content determination of N-(E)-p-coumaroyltyrosine in leaves of Abrus cantoniensis,and to optimize its purification technology. METHODS:HPLC method was adopted for the content determination of N-(E)-p-coumaroyltyrosine in A. cantoniensis. The determination was performed on Hypersil BDS C18 column with mobile phase consisted of 0.1% formic acid water(A)-methanol(B)(gradient elution)at a flow rate of 1.0 m L/min. The column temperature was set at 25 ℃. The detection wavelength was set at 300 nm,and sample size was 10 μL. Using polyamide resin as material,the yield of N-(E)-p-coumaroyltyrosine as indicators,single factor test was used to optimize the purification technology of N-(E)-pcoumaroyltyrosine,such as concentration,sample size,stationary adsorption time. RESULTS:The linear range was 2.575-51.50μg(r=0.999 9)for N-(E)-p-coumaroyl-tyrosine. The limit of quantification was 0.000 618 μg,and the detection limit was 0.000129 μg. RSDs of precision,stability and reproducibility tests were all lower than 3%. The recoveries were 97.04%-102.43%(RSD=2.06%,n=6). The optimal purification technology was as follows:the concentration of the sample solution was 0.04 g/mL(by the leaves of A. cantoniensis);sample capacity 50 mL;the sample flow rate was 1.0 m L/min;the stationary adsorption time was 20 min;the eluting solvents were ammonia containing water(containing 0.1% acetic acid),20% ethanol(containing0.1% acetic acid)and ammonia(p H 10). Average yield was 98.94%,average dry paste content was 61.17 mg/g,and average dry paste purity was 19.73% by optimal purification technology. CONCLUSIONS:Established method is simple,accurate and stable.The optimized technology is stable and feasible.
OBJECTIVE:To establish a method for content determination of N-(E)-p-coumaroyltyrosine in leaves of Abrus cantoniensis,and to optimize its purification technology. METHODS:HPLC method was adopted for the content determination of N-(E)-p-coumaroyltyrosine in A. cantoniensis. The determination was performed on Hypersil BDS C18 column with mobile phase consisted of 0.1% formic acid water(A)-methanol(B)(gradient elution)at a flow rate of 1.0 m L/min. The column temperature was set at 25 ℃. The detection wavelength was set at 300 nm,and sample size was 10 μL. Using polyamide resin as material,the yield of N-(E)-p-coumaroyltyrosine as indicators,single factor test was used to optimize the purification technology of N-(E)-pcoumaroyltyrosine,such as concentration,sample size,stationary adsorption time. RESULTS:The linear range was 2.575-51.50μg(r=0.999 9)for N-(E)-p-coumaroyl-tyrosine. The limit of quantification was 0.000 618 μg,and the detection limit was 0.000129 μg. RSDs of precision,stability and reproducibility tests were all lower than 3%. The recoveries were 97.04%-102.43%(RSD=2.06%,n=6). The optimal purification technology was as follows:the concentration of the sample solution was 0.04 g/mL(by the leaves of A. cantoniensis);sample capacity 50 mL;the sample flow rate was 1.0 m L/min;the stationary adsorption time was 20 min;the eluting solvents were ammonia containing water(containing 0.1% acetic acid),20% ethanol(containing0.1% acetic acid)and ammonia(p H 10). Average yield was 98.94%,average dry paste content was 61.17 mg/g,and average dry paste purity was 19.73% by optimal purification technology. CONCLUSIONS:Established method is simple,accurate and stable.The optimized technology is stable and feasible.
引文
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[2]SANBONGI C,OSAKABE N,NATSUME M,et al.Antioxidative polyphenols isolated from theobroma cacao[J].J Agric Food Chem,1998,46(2):454-457.
[3]SENDKER J,B?KER I,LENGERS I,et al.Phytochemical characterization of low molecular weight constituents from marshmallow roots(althaea officinalis)and inhibiting effects of the aqueous extract on human hyaluronidase-1[J].J Nat Prod,2017,80(2):290-297.
[4]ZOLFAGHARI B,BARILE E,CAPASSO R,et al.The sapogenin atroviolacegenin and its diglycoside atroviolaceoside from Allium atroviolaceum[J].J Nat Prod,2006,69(2):191-195.
[5]HENSEL A,DETERS AM,MüLLER G,et al.Occurrence of N-phenylpropenoyl-L-amino acid amides in different herbal drugs and their influence on human keratinocytes,on human liver cells and on adhesion of Helicobacter pylori to the human stomach[J].Planta Medi,2007,73(2):142-150.
[6]OSAKABE N.Antioxidative polyphenolic substances in cacao liquor[J].Acs Symposium,2000.DOI:10.1021/bk-2000-0754.ch011.
[7]MOKHTAR M,SOUKUP J,DONATO P,et al.Determination of the polyphenolic content of a Capsicum annuum L.extract by liquid chromatography coupled to photodiode array and mass spectrometry detection and evaluation of its biological activity[J].J Sep Sci,2015,38(2):171-178.
[8]HU XL,LIN J,LV XY,et al.Synthesis and biological evaluation of clovamide analogues as potent antineuroinflammatory agents in vitro and in vivo[J].Eur J Med Chem,2018.DOI:10.1016/j.ejmech.2018.03.081.
[9]袁旭江,张平,李春阳,等.毛鸡骨草叶中一个新的吐昔酰胺类成分[J].药学学报,2016,51(10):1595-1599.
[10]马琼.木棉花化学成分研究及活性部位的抗糖尿病作用[D].呼和浩特:内蒙古大学,2016.
[11]国家药典委员会.中华人民共和国药典:三部[S].北京:中国医药科技出版社,2015:831.
[12]袁旭江,李春阳,张平.不同品种鸡骨草叶中鸡骨草甲素含量比较[J].现代中药研究与实践,2014,28(4):19-22.